A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (111In-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules.
The antigen specificity of monoclonal antibodies is a powerful attribute that allows the site-specific in vivo delivery of payloads, including chemotherapeutic drugs and radionuclides (Wu, A. M.; Senter, P. D. (2005) Nat Biotechnol., 23:1137). To date, only two radioimmunotherapeutic agents have received marketing approval, and both feature murine monoclonal antibodies targeting the CD20 receptor for treatment of lymphoma (Boswell, C. A., et al (2007) Nucl Med Biol, 34:757). BEXXAR® (tositumomab) incorporates the β-emitting iodine radionuclide, 131I, attached via tyrosine residues. ZEVALIN® (ibritumomab tiuxetan) is administered with the β-emitting yttrium radionuclide, 90Y, attached via tiuxetan, an analog of diethylenetriamine pentaacetic acid (DTPA), through lysine residues. This labeling strategy is analogous to the complexation of the indium radionuclide, 111In, by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Various methods of radiolabeling antibodies are known, including: (A) non-residualizing, oxidative radioiodination of tyrosines, (B) residualizing, lysine modification with radiometal chelate, (C) residualizing, lysine modification with charged iodinated groups (Vaidyanathan, G.; et al, (2001) Bioconjug Chem., 12:428; Shankar, S. et al, (2003) Bioconjug Chem., 14:331), and (D) lysine modification with DOTA-SIB (Vaidyanathan et al (2012) Bioorg. & Med. Chem. 20:6929-6939).
Beyond their clinical utility, radioimmunoconjugates are also useful as tools in translational research for studying conventional, non-radioactive antibody therapeutics (Boswell, C. A., et al (2012) Aaps J., 14:612). Confirmation of target localization, screening for off-target uptake, and receptor occupancy studies (by means of dose escalation) may all be facilitated by the use of radiolabeled antibodies. The available in vivo modalities include non-invasive small animal imaging, whole-body autoradiography, and invasive biodistribution studies. In addition to 131I, 125I is commonly used for such studies, with the latter having the advantages of a roughly tenfold lower γ (gamma) energy, the absence of a β (beta) particle emission, and a much longer decay half-life (Wilbur, D. S. (1992) Bioconjug Chem, 3:433). See Table 1. Single photon emission computed tomography (SPECT) imaging may be performed with 123I, 131I, and 125I, the latter being limited to preclinical small animal cameras. Positron emission tomography (PET) with 124I is also feasible, although the highly energetic emissions limit the image quality (Williams, S. P. (2012) AAPS J. 14(3):389-99. doi: 10.1208/s12248-012-9348-3. Epub 2012 Mar. 31.
Tissue distribution studies of protein therapeutics can be conducted using molecular probes and molecular imaging.
Labeling of antibodies with radiometals results in a different cellular distribution of radioactivity relative to traditional tyrosine-based radiohalogenation (Shih, L. B., et al (1994) J Nucl Med, 35:899). For both labeling methods, antibodies undergo receptor-mediated endocytosis and lysosomal degradation. However, cellular efflux of the radiolabel with its covalently associated amino acid does not occur for radiometal-labeled antibodies, see FIGS. 1A and 1B (Rogers, B. E., et al (1995) Cancer Res., 55:5714s; Vaidyanathan et al (2012) Bioorg. & Med. Chem. 20:6929-6939). Antibodies labeled with 125I through tyrosine residues undergo (1) receptor-mediated endocytosis, (2) lysosomal degradation and (3) diffusion of [125I]-iodotyrosine out of the cell. Steps (1) and (2) also occur for antibodies labeled with 111In-DOTA through lysine residues; however, step (3) is greatly diminished due to the poor membrane diffusion of the radiolabeled catabolite, 111In-DOTA-lysine. Unlike [125I]-iodotyrosine, which diffuses out of the cell following proteolysis, In-DOTA-lysine is too charged and polar to easily cross the plasma membrane and is therefore intracellularly trapped and referred to as a residualizing label. The relatively short decay half-life of 2.8 days for 111In makes long-term preclinical studies problematic particularly for labeled antibodies with pharmacokinetic half-lives on the order of 1-2 weeks. Another consideration is that the γ energy of 125I is nearly ten-fold lower relative to 111In, with lower energy emissions often associated with superior autoradiographic image quality and lower radiation exposure to workers. A residualizing iodine probe would combine the long decay half-life and low energy of 125I with the superior tumor accretion of radiometals, while providing a facile translational route to clinical imaging via 123I or 124I, or radioimmunotherapy via 131I (Milenic et al (2004) Nat. Rev. Drug Discov. 3:488-499).
TABLE 1Overview of common halogen radionuclides.Emission† (Energy,Physical DecayDiagnostic and/orNuclidekeV)t1/2 (d)Therapeutic Uses123Iγ (159)0.5SPECT imaging124Iγ (603),4.2PET imagingβ+ (831)125Iγ (35)60preclinical SPECT131Iγ (365),8.0radioimmunotherapy,β− (182)SPECT211Atα (5867),0.3radioimmunotherapyγ (687)†Values correspond to the most abundant γ emissions and the mean α/β energies, respectively.
Significant effort has been made to derive strategies for labeling antibodies with iodine such that residualization occurs in a similar manner as for radiometals. This reflects, in part, the wide availability of iodine radionuclides with diverse nuclear properties, in terms of both decay half-lives and energies (Table 1), and an abundant knowledge of halogen radiochemistry (Wilbur, D. S. (1992) Bioconjug Chem, 3:433). To date, strategies used to achieve this goal include the use of various combinations of (i) nonmetabolizable carbohydrates, (ii) nonmetabolizable peptide adducts, and/or (iii) synthetically derived molecules containing charged moieties. The carbohydrate derivative dilactitol-125I-tyramine is a member of the (i) first class of residualizing radioiodine probes (Thorpe, S. R., et al (1993) Faseb J., 7:399). However, the use of carbohydrates may produce unwanted behavior, as pendant sugar groups are important for binding of antibodies to Fc receptors and other critical functions. Representing the (ii) second class is the residualizing peptide, IMP-R4 (MCC-Lys(MCC)-Lys(X)-D-Tyr-D-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-DTPA (Stein, R., et al (2003) Cancer Res., 63:111). This approach relies on a synthetic peptide that is conjugated with the chelate DTPA, whose charge imparts residualizing properties (Govindan, S. V., et al (1999) Bioconjug Chem., 10:231). Examples of the (iii) third class of charged synthetic molecules, many of which involve lengthy synthetic routes, include the use of organostannanes (Vaidyanathan, G., et al (2001) Bioconjug Chem., 12:428; Shankar, S.; Vaidyanathan, G., et al (2003) Bioconjug Chem, 14:331; Vaidyanathan et al (2012) Bioorg. & Med. Chem. 20:6929-6939). In this direction, a shelf-stable intermediate compound that is readily attainable via synthetic organic chemistry and avoids the use of peptide or carbohydrate moieties, or lengthy synthetic routes, would be useful for radiohalogen-labeling proteins.